The objective of this project is to provide an answer to a major question concerning the sarcoplasmic reticulum, namely what is the mechanism of calcium transport through the phospholipid bilayer. The role of protein through the membrane will be investigated in which multiple subunit aggregates of the Ca ion-activated ATPase mediate calcium transport. Protein aggregate formation will be determined by chemical crosslinking followed by analysis of crosslinked proteins by sodium dodecyl sulfate gel electrophoresis, and freeze-fracturing electron microscopy. These methods will be used to look for changes in the oligomeric state of membrane proteins as the Ca 2 ion-activated ATPase is interconverted between the different enzymatic states that accompany calcium accumulation. To determine the role of Calsequestrin and Calcium Binding Protein, possible interactions between these minor protein components and the Ca ion-activated ATPase in terminal cisternae-derived sarcoplasmic reticulum vesicles will be investigated. The calcium pumping activity of cardiac sarcoplasmic reticulum can be stimulated 3-fold by the action of a cAMP-dependent protein kinase. To determine whether this stimulation is mediated via protein-protein interactions, the relationship between the many proteins in cardiac sarcoplasmic reticulum and the Ca 2 ion-activated ATPase will be investigated as the extent of kinase-mediated phosphorylation is varied. In addition interactions between the protein phosphorylated by the kinase, Phospholamban, and the Ca 2 ion-activated ATPase will be looked for to determine the mechanism by which this phosphorylation can increase Ca 2 ion-activated ATPase activity. The goal of this project is to show whether ionic channels in membranes can be regulated by changes in monomer-oligomer equilibria of membrane proteins.